CN106461515A - Miniature serial sectioning microtome for block-face imaging - Google Patents
Miniature serial sectioning microtome for block-face imaging Download PDFInfo
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- CN106461515A CN106461515A CN201580025001.4A CN201580025001A CN106461515A CN 106461515 A CN106461515 A CN 106461515A CN 201580025001 A CN201580025001 A CN 201580025001A CN 106461515 A CN106461515 A CN 106461515A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/20—Means for supporting or positioning the objects or the material; Means for adjusting diaphragms or lenses associated with the support
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/22—Optical or photographic arrangements associated with the tube
- H01J37/222—Image processing arrangements associated with the tube
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/30—Electron-beam or ion-beam tubes for localised treatment of objects
- H01J37/317—Electron-beam or ion-beam tubes for localised treatment of objects for changing properties of the objects or for applying thin layers thereon, e.g. for ion implantation
- H01J37/3178—Electron-beam or ion-beam tubes for localised treatment of objects for changing properties of the objects or for applying thin layers thereon, e.g. for ion implantation for applying thin layers on objects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
- G01N2001/061—Blade details
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
- G01N2001/065—Drive details
- G01N2001/066—Drive details electric
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
- H01J2237/202—Movement
- H01J2237/20207—Tilt
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
- H01J2237/202—Movement
- H01J2237/20214—Rotation
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
- H01J2237/206—Modifying objects while observing
- H01J2237/2067—Surface alteration
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/22—Treatment of data
- H01J2237/226—Image reconstruction
Abstract
The present disclosure is directed to embodiments of microtome devices and methods of their use. In some embodiments, a microtome can be mounted on the built-in stage of a scanning electron microscope and used to perform serial block-face scanning electron microscopy. In some cases, a microtome installed in a scanning electron microscope can cut the sample at a location off the electron beam axis of the scanning electron microscope. In some cases, a microtome can include a capacitive sensor which can measure the location of a blade of the microtome, and the microtome can be computer-controlled by program implemented in MATLAB.
Description
Cross-Reference to Related Applications
This application claims the U.S. Provisional Application No.61/991 that on May 12nd, 2014 submits to, 929 rights and interests, the described U.S.
Provisional application is hereby incorporated herein by.
Technical field
Present disclosure is related to microtome (microtome) equipment and its using method, relates in particular to be designed to use
Section machine equipment in block face imaging applications (including the imaging based on optics or electronics).
Background technology
A kind of application of block face imaging technique is serial block Surface scan electron microscopy (sometimes referred to as SBEM, SBSEM
And/or SBFSEM), it refers to produce multiple two dimensional images of sample on continuous level along third dimension, thus produces and closes
Process in the data of the three dimensional structure of sample.SBSEM technology can be used for studying many different types of biological samples, and
It is frequently used for studying cerebral tissue.It is used in particular for collecting high-resolution anatomical data, such as when the aixs cylinder in mapping brain and
When neuronal circuit connects.One SBSEM process is included using scanning electron microscope (SEM) come by collecting secondary and carrying on the back scattered
Radio obtains two dimensional image, and is removed between successive images using microtome (sometimes referred to as " ultramicrotome ")
Very thin (for example, in tens nanometer range) at sample top part.By the vacuum chamber in scanning electron microscope
Middle installation microtome, can make this process more effective.Previous microtome has various shortcomings, as further described herein.Cause
This, need the improvement of microtome technology.
Content of the invention
In some embodiments, a kind of microtome of a thin part at the top for removing sample comprises：Base
Plate；Pedestal, is connected to substrate so that pedestal can move to cutting position from image space, and wherein have can be upper for pedestal
The exposed surface of sample is installed in face；Blade, is connected to substrate so that when pedestal is in image space, can pass through vertical
On the direction of the exposed surface of pedestal, moving blade to be adjusting the blade position with respect to substrate, to selectively change blade
The distance between with substrate, wherein cutting position than image space closer to blade.
In certain embodiments, microtome is arranged on the fluorescence microscopy being configured to the surface imaging to sample using camera
On mirror.In certain embodiments, microtome be arranged on be configured to the surface imaging to sample using camera cathodoluminescence show
On micro mirror.In certain embodiments, microtome is arranged on the photoemission being configured to the surface imaging to sample using camera
On ultramicroscope.In certain embodiments, blade is arranged on the linear actuatorss of the first computer controls being connected to substrate
On.In certain embodiments, pedestal is arranged on bar, and described bar is connected to substrate by pivot bearings.In certain embodiments,
By being connected to the actuating of the linear actuatorss that the second computer of substrate controls, pedestal can rotate around pivot bearings.
In certain embodiments, the first actuator is configured to receive control signal with the motion of guiding blade, microtome
Comprise further：Sensor, described sensor is connected to the first actuator and is configured to produce instruction with respect to substrate
The output signal of blade position；And, computer program, it is configured to receive the output signal from sensor, at least partly
Ground produces control signal based on the output signal from sensor, and transmits control signals to the first actuator.
In certain embodiments, microtome comprises the sample being positioned on pedestal further.In some embodiments it is possible to
The operating distance between the sample of scanning electron microscope and pole piece is selected from the scope of available work distance.In some enforcements
In example, voltage is applied to sample.In certain embodiments, sample and microtome electrically insulate.In certain embodiments, blade is
The vibration diamond blade that piezoelectricity controls.In certain embodiments, microtome comprises further including processor and memorizer
Computing device, the multiple images that memory storage is used for combined sample are referred to the computer-readable creating the three dimensional representation of sample
Order.
In some embodiments, a kind of microtome being configured to be arranged in scanning electron microscope comprises：Connection
To the blade of actuator, wherein actuator is connected to the platform of scanning electron microscope so that actuator can be parallel to sweeping
With respect to platform moving blade on the direction of the beam axis retouching ultramicroscope, and wherein actuator is configured to receive control
Signal is with the motion of guiding blade；Sensor, is connected to actuator and is configured to produce the blade with respect to substrate for the instruction
The output signal of position；And, computer program, it is configured to receive the output signal from sensor, at least part of ground
Produce control signal in the output signal from sensor, and transmit control signals to actuator.
In certain embodiments, microtome comprises pedestal further, and described pedestal is connected to platform so that pedestal can be from
Image space on beam axis moves to the cutting position leaving beam axis, wherein cutting position than image space closer to knife
Piece.
In some embodiments, a kind of method comprises：By Sample location on the microtome in scanning electron microscope
Image space at, wherein image space is on the beam axis of scanning electron microscope；The first exposed surface imaging to sample；
The height of the blade of setting microtome；Sample is moved to cutting position from image space, wherein cutting position compares image space
Closer to blade and not on beam axis；Mobile example passes through blade, to remove a part for sample and to expose the of sample
Two exposed surfaces；Sample is moved to image space；And the second exposed surface imaging to sample.
In certain embodiments, after the height further contained in setting blade for the method, keep knife under the feedback control
The height of piece.In certain embodiments, before method is further contained in being imaged to the second exposed surface, by scanning electron microscopy
The Electron Beam Focusing of mirror is at the second exposed surface.In certain embodiments, method is further contained in the second exposed surface
Before imaging, along beam axial adjustment sample.In certain embodiments, method is further contained in along beam axial adjustment sample
Afterwards, by the Electron Beam Focusing of scanning electron microscope at the second exposed surface.
In certain embodiments, the action to the first exposed surface imaging of sample comprises to come using scanning electron microscope
Capture multiple composition images of the first exposed surface, and by multiple composition image mosaic together to form the first exposed surface
Composograph.In certain embodiments, method comprises to make microtome tilt so that beam axis is not orthogonal to sample further
Exposed surface.In certain embodiments, method comprises to make microtome rotate further.In certain embodiments, method is further
Comprise with polystyrene foam plastics channel washer cleaning blade.In certain embodiments, microtome is arranged on halfpace, and
And halfpace is arranged on the built-in platform of scanning electron microscope.
The several embodiments being carried out by reference to accompanying drawing described in detail below, disclosed technology foregoing and other
Feature and advantage will become apparent from.
Brief description
Fig. 1 is the top perspective view of example slice machine.
Fig. 2A to Fig. 2 C exemplifies the part of the microtome of Fig. 1 with bigger ratio.
Fig. 2 D diagram is using the method for the microtome of Fig. 1.
Fig. 3 A to Fig. 3 O is the several continuous scanning of the tissue sample producing during serial block Surface scan electron microscopy
The image of the calculating difference between electron microscope image, and the continuous scanning electron microscope image of diagram tissue sample.
Fig. 4 A is to illustrate when moving tissue sample through blade several times, the electric capacity of the vertical position of measurement microtome blade
The curve chart of the output of sensor.
Fig. 4 B is the curve chart of the data sequence of Fig. 4 A illustrating to overlap each other.
Fig. 5 is the diagram of the position illustrating multiple composition scanning electron microscope images.
Fig. 6 A to Fig. 6 C is the scanning electron microscope image of the same sample shooting under different operating distance.
Fig. 7 is that wherein voltage has been applied to electrically insulate the side view of the microtome of sample.
Fig. 8 A to Fig. 8 C diagram controls sample using the microtome (microtome of such as Fig. 1) in scanning electron microscope
The ability tilting.
Fig. 9 A to Fig. 9 E is the different views of another exemplary microtome.
Figure 10 A and Figure 10 B illustrate respectively the tissue slice of 15 nanometer thin using disclosed microtome cutting XY and
XZ view.
Figure 11 is the axonometric chart of another example slice machine.
Specific embodiment
Microtome as herein described can be used for various Microbeam Analysis Techniques, is such as used for scanning electron microscopy, is based on
(optical, such as fluorescence) microscopy of light, cathodoluminescence microscopy, or the combination for these technology.For example, sample can
To be arranged on microtome as described below, it is (for example, glimmering that this microtome can be directly installed on common (optics) based on light
Light) under microscopical object lens.These embodiments can be in atmospheric conditions and in the case of not having scanning electron microscope
Operation.These technology can utilize various fluorescence microscopy (for example, Multiphoton microscopy, super-resolution as known in the art
Rate microscopy etc.).
In certain embodiments, microtome as herein described can be used for optics and/or cathodoluminescence microscopy and scanning
Electron microscopy, and can capture and store the image of continuous exposed surface.Image can also be carried out with numerical analyses and weight
Group, to provide the 3-D view of tissue under the microscopic resolution of height or to represent, for example, with the nerve of determination or mapping object
First loop.In some embodiments, this can be by using such as fluorescence detector, camera or cmos sensor via scanning
Observation port in the inner vacuum vessel of ultramicroscope is imaged to realize to sample.In some cases, can be in SBEM process
Period shoots cryptoscope and/or cathodoluminescence image to each exposed surface of sample so that each scanning electron microscope diagram
As having corresponding cryptoscope and/or cathodoluminescence image.Part reference described below is during SBEM using microtome
Carry out, but present disclosure is commonly available in any application using microtome.
Microtome as herein described can be used for studying any one of various types of specimen materials and for various
Purpose, including the inspection of the tissue specimen for cytobiology, carcinobiology and/or immunological investigation.Microtome is special
It is applied to the biological tissue samples of plastic embedding, the cerebral tissue of such as plastic embedding.
Scanning electron microscopy allows the very high-resolution imaging of sample, which results in its making in various fields
With.Many business scanning electron microscopes comprise internal come-at-able vacuum chamber, and sample is positioned in this vacuum chamber and is used for leading to
Cross microscope imaging.By the door of microscope side Lai close, door is turned off to provide gas-tight seal to chamber internal chamber.Interior
Then portion's chamber is drained air to reduce the interference to imaging process (for example, the scattering to electron beam by gas molecule).
Many scanning electron microscopes are also contained in the platform that to be imaged sample is located above, and along or with respect to
Three axle (for example, the z-axis with microscopical electron beam alignment, and the x-axis perpendicular to z-axis and y-axis) linear translation stage.Flat
Platform can also be around z-axis rotation and around the inclination of at least one of x-axis and y-axis so that the face of platform can be with respect to electronics
Shu Xuanzhuan and inclination.In some ultramicroscope, platform can rotate 360 degrees around z-axis and around in x-axis and y-axis
Individual inclination in the range of 90 °.Can be used for being aligned sample under electron beam around z-axis rotary microtome.
In the research of material particularly biological tissue, the data of the three dimensional structure of description sample is possibly highly desirable to
's.In order to obtain three-dimensional data, have been developed for may be mounted at the microtome of the vacuum interior of scanning electron microscope.Sample
It is arranged on microtome and be oriented to by microscope imaging.Once the exposed surface imaging to sample, then using microtome
To remove the very thin part at sample top, thus exposing new surface to be imaged.This process can repeat, until shooting
The image of requirement, or until being imaged to the thickness needed for sample.The a series of images so shooting can illustrate sample
The three dimensional structure of product.
Known section machine equipment has various shortcomings.As an embodiment it is known that microtome make blade movement wear
Cross sample to remove the thin part at sample top.Because sample is located immediately at below microscopical detector and pole piece,
Fragment from sample sections may contact or adhere to the pole piece of detector and/or electron beam, thus the behaviour of interference microscope
Make.In some cases, there is pressure differential in the porch of the pole piece of ultramicroscope, thus increased fragment to enter electron beam
Chance and exacerbate this problem.Additionally, in order to allow blade in the space of sample top movement it is necessary in sample and detection
Enough spaces are provided between device, thus reduce the scope of possible operating distance.Especially, which has limited and can reduce work
The degree of distance, this transfers to limit the quality of the data that can obtain.For example, reduce the work between sample and detector
Distance can improve the signal to noise ratio in the data obtained.
As another embodiment it is known that the section that can obtain from the Gatan company of Pennsylvania Warrendale
Machine is installed on the steel casement door of customization.In order to install this microtome in scanning electron microscope it is necessary to remove microscopical former
Beginning platform and door, and the steel casement door of customization is arranged on home position.This makes microtome relatively heavy and is difficult to install.This
Outward, this microtome only allows sample to translate along x-axis, y-axis and z-axis, but does not allow what the built-in platform of sample image allowed equally to incline
Tiltedly or rotation.Additionally, the translation that this equipment provides along x-axis and y-axis is not real straight line.Rather, it should be appreciated that Gatan
Microtome provide along big parabolic arc translation so that translation close to but be not exclusively straight line.Known microtome has
Other shortcomings, this will be described in greater detail below.
Section machine equipment as herein described allows in cutting sample at the position that electron beam axis remove, thus reduce being derived from
The chance of the interference of fragment, and allow imaging in the range of bigger operating distance.By allowing operating distance than known
Reduce and provide larger range of operating distance for selection in equipment further, this is probably favourable.As herein described
Section machine equipment can also be relatively light and therefore be more easily installed, and is designed in some embodiments pacify
It is contained on the built-in platform of various scanning electron microscopes so that can be along the real rectilinear translation of three axles (at many lis
In the range of rice) and position and the orientation of samples is finely controlled around two rotary shafts.Microtome as herein described can be from
Very thin part is reproducibly cut at sample top.Microtome as herein described has the advantages that extra, and this will below more
Describe in detail.
Fig. 1 and Fig. 2A to Fig. 2 C illustrates the example slice machine 100 being suitable in scanning electron microscope.When wherein
During using microtome 100, microtome 100 is arranged on the platform of scanning electron microscope, to control the translation of microtome 100
And rotation.Microtome 100 can be installed with microscopical platform with flushing, or can be connected to platform and pass through multiple posts
It is spaced apart with platform.Microtome 100 can be used in combination with any one in various commercially available scanning electron microscopes, and these are swept
Retouch ultramicroscope there is sufficiently large internal chamber and (microtome 100 exemplary is provided below to be adapted to microtome wherein
Overall dimension).For example, microtome 100 can be used for available commercially from Hillsboro, the NOVA NANOSEM of FEI Co. of OR
50 series of scans ultramicroscope.A kind of suitable scanning electron microscope is FEI NanoSEM 450 equipment.
Microtome 100 includes the substrate 102 being connected to platform.Support 106 is connected to substrate 102 and includes vertical surface
108, linear actuatorss 110 are installed to this vertical surface 108.Can be using any one in various suitable linear actuatorss.
As embodiment, the N-661 micro linear platform with NEXACT driver is (before available commercially from Physik
), and its substitute products linear piezoelectric platform LPS-45 is (at present available commercially from Physik Instrumente
Instrumente), in conjunction with its correlation control unit unit, it is suitable linear actuatorss.Linear actuatorss 110 can produce it
Platform 110B is with respect to the translation of its main body 110A.
The H-shaped installation elements 112 with two vertical posts 112A and central cross bar 112B are installed to the exposure of platform 110B
Surface.Blade support member 114 including vertical section 114A and two horizontal protruded arm 114B is installed to central cross bar 112B.Arm
114B is spaced apart from each other and extends so that blade 116 may be mounted at its terminal away from vertical section 114A in parallel with each other
Between.Suitable blade is well known in the art.The blade being particularly well-suited to microtome 100 includes the vibration of piezoelectricity control
Diamond blade, such as in the Minimal Compression of Ultrathin Sections with of Studer et al.
Use of an Oscillating Diamond Knife, Journal of Microscopy, volume 197, part 1, the
Blade described in 94-100 page (in January, 2000).Various specially suitable commercially available blades are provided by DiATOME U.S.
As shown in more detail in Fig. 2 B and Fig. 2 C, blade 116 is arranged between the terminal of arm 114B, and screw 144
Through arm 114B and tighten so that blade 116 is sandwiched in arm 114B.Piezo-activator 146 is connected to knife by screw 148
Vertical section 114A of piece support member 114, screw 148 is by the spiral shell on the end of the screw 148 relative with piezo-activator 146
Mother is fixed to blade support member 114.Blade support member 114 can be connected to bender element 150, and blade is propped up by bender element 150
Support member 114 is connected to the remainder of microtome 100.Bender element can be to have sufficiently flexible narrow, pliable and tough metal
Element is so that the actuating of piezo-activator 146 causes blade support member 114 to bend with bender element 150 vibrates back and forth.Bending
Element 150 can have the width of 1mm.Piezo-activator 146 may be configured to the blade support member on bender element 150
114 resonant frequency vibration.A kind of suitable piezo-activator is the fine vacuum compatibility that can obtain from PI Ceramic
PD080.31 through hole piezo-activator, and the signal of the correlation that can obtain from Physik Instrumente amplifies and regulation sets
Standby.
Microtome 100 also includes sensor cluster 118 (Fig. 1), and it is included for being held in place by sensor
The capacitance sensor of sensor pincers 120, inclusion axle 122 and capacitive sensor modules 124, and the target plate being connected to substrate 102
126.Various suitable capacitance sensors are commercially available, and one of embodiment can obtain from Physik Instrumente
D-510PISECA capacitance sensor and related signal regulating equipment, it can be measured with nanometer resolution.Capacitance sensing
Device can provide the distance between the exposed bottom surface of instruction capacitive sensor modules 124 and the exposed upper surface of target plate 126
Output signal.Sensor cluster 118 is connected to vertical posts 112A of installation elements 112, therefore, is knowing each of microtome 100
In the case of planting the size of part, output signal can indicate the height that blade 116 is with respect to sample 136.
Come self capacitance sensor output signal can be adjusted by data and/or acquisition system such as available commercially from
18 of National Instruments Corporation, 625kS/s, NI USB-6289M series multifunctional DAQ system
To run, and to run in software program such as available commercially from the program implemented in the MATLAB of Mathworks company.Journey
Sequence may be configured to receive the output signal of self capacitance sensor, receives input from user (for example, blade 116
Required height), and provide output signal with the motion of control platform 110B to actuator 110.Receiving instruction blade
116 will be moved to after the input of required height, and program can be with the specified altitude assignment of calculating platform 110B, thus by blade 116
It is positioned at required height.Then, program can send the signal including the order moving to specified altitude assignment to actuator 110.
Program can monitor the signal of self capacitance sensor further, to determine whether blade is actually moved to and is maintained at specify height
Degree.
If the actual blade height of signal designation carrying out self capacitance sensor is less than required blade height, program is permissible
Send new signal to actuator 110, this new signal includes moving to the order of the new high degree more than specified altitude assignment.If from electricity
The actual blade height of signal designation holding sensor is higher than required blade height, then program can send newly to actuator 110
Signal, this new signal includes moving to the order of the new high degree less than specified altitude assignment.This process can continuously be run, thus being based on
Blade 116 is maintained at the closed control loop (or feedback circuit) of required height by the signal creation carrying out self capacitance sensor
(for example, using pid control circuit feedback mechanism).This can help improve the precision of microtome system, including microtome system
Remove precision and the uniformity of material from the top of sample 136.
Microtome also includes：Two pivot support elements 128, it is spaced apart from each other and is connected to substrate 102；Pivotal axis
Hold 130, it is installed to pivot support element 128 and crosses between pivot support element 128；And, rotatable bar 132,
It is arranged in pivot bearings 130 for the axis rotation around pivot bearings.Pivot bearings 130 include：Standing part, its
It is rigidly connected to pivot support element 128 and supported by pivot support element 128；And, rotatable portion, it is with respect to rigidity
It is connected to bar 132 and the standing part of support bar 132 is freely rotatable.Various suitably commercially available pivot bearings can be used,
Such as available commercially from the 6016-800 pivot bearings of Riverhawk company.At the top of rotatable bar 132, microtome 100 includes
Can be in the pedestal 134 of sample 136 to be studied installed above.Suitable pedestal includes the sample for scanning electron microscope
Frame, such as available commercially from the EMS aluminum sample stake 75638-10 of Electron Microscopy Sciences.
Microtome 100 also includes the pincers 138 being connected to substrate 102, and is clamped in the linear actuatorss 140 in pincers 138.
Can be using any one in various commercially available linear actuatorss, one of specially suitable actuator is can be from Physik
N-381NEXACT linear actuatorss, manipulator, piezoelectricity stepper product and correlation control unit that Instrumente obtains.Linearly
Actuator 140 be connected to rotatable bar 132 so that actuator 140 can with control bar 132 around pivot bearings 130 rotation.More
Specifically, actuator 140 terminates at ball bearing, this ball bearing with positioned at bar 132 within sapphire pad engage.Lan Bao
Stone pad can provide the very flat and durable surface for engaging actuator 140.Suitable sapphire pad include by
The sapphire pad that Thorlabs company is sold with model P25SK2-Polaris-Kl.
In some cases, bar 132 can be biased (for example, bar 132 can be increased) to revolve around pivot bearings 130
Turn away from blade 116.Actuator 140 can push sapphire pad, with actuator lever 132, sample 136 is moved towards blade 116.?
In one specific embodiments, bar 132 can be increased so that it is biased to the rotation around about 1 degree of pivot bearings 130
Shelve be than scanning electron microscope electron beam further from blade 116.Actuator 140 can engage sapphire pad so that bar
132 towards blade 116 rotate 1 degree so that sample 136 be positioned at scanning electron microscope electron beam in.Actuator 140 is permissible
Engage sapphire pad so that bar 132 rotates about 10 degree so that sample 136 is positioned in the region of blade 116.
In order to prevent actuator 140 from so that sample 136 is rotated up too far in the side of blade 116 and actuator 110, microtome
Also include pivoting detent part 142, this pivoting detent part 142 is connected to pivot support element 128 and in bar 132 and actuator
Extend between 110.Microtome 100 can also include weight 152 (such as bloom), to increase extra weight to actuator 110
To stablize blade 116, and to keep the position of blade 116 when sample 136 translates across blade 116.
Actuator 140 can be used for mobile example 136 and passes through blade 116, for example, to remove the exposed surface of sample 136
A part.For example, above-mentioned MATLAB program may be configured to from user receive defeated with regard to the motion needed for sample 136
Enter, and provide output signal with the motion of control bar 132 to actuator 140 and therefore control the motion of sample 136.Connecing
Receive instruction sample 136 will be moved to after the input of desired position, program can calculate Sample location in required position
The specified location of the actuator 140 put.Then, program can send, to actuator 140, the order including moving to specified location
Signal.
In some cases, sample to be studied can be embedded in plastics (for example, epoxy resin) to promote sample
Accurately cut and be imaged.Prepare for the sample in the microtome of serial block Surface scan electron microscopy method in the art
It is known.As an embodiment of this method, tissue sample to be studied can be cut into certain size, Ran Hou
In embedded liquid epoxies in mould, afterwards can cured epoxy resin in an oven.Sample and the epoxy being embedded with sample
Resin can cut out from mould and be glued to it by the base being supported on microtome or pedestal.It is, for example possible to use it is embedding
Enter to have the identical epoxy resin of sample that sample is glued to pedestal.
Once sample is glued to pedestal, it is possible to use business ultramicrotome removes the unwanted part of sample, by
This produces truncated pyramid or the trapezoidal prism shape sample that tissue is exposed to its side.These shapes provide relative stiffness and stablize
Sample, it more accurately and consistently can cut than the sample of other shapes.Then sample can be coated in such as gold
In conductive material, so that by the side ground of sample to pedestal, thus allowing dissipation electronics preventing in imaging phase from sample
Between electrostatic charge accumulation.Then removing coating can be removed from the top of sample, be come in scanning electron with the upper surface of exposed sample
It is imaged in microscope.
Fig. 2A illustrates the zoomed-in view of the part of microtome 100, and wherein sample 136 is located at below pole piece and scanning electron shows
On the beam axis of micro mirror.Fig. 2 B is shown in MATLAB program and sends order to actuator 140, and actuator 140 passes through to activate
Sample 136 is moved towards blade 116 and removes below pole piece and come away from the beam axis of scanning electron microscope by bar 132
After this order, the zoomed-in view of the part of the microtome of Fig. 2A.
Microtome as herein described can reproducibly cut very thin part from the top of sample.Microtome can be cut
Cut the section that thickness is equal to the resolution of capacitance sensor, this thickness can be less than 10 nanometers.It is used for scanning electron in microtome
In the case of in microscope, slice thickness is limited by the electron dose being applied to the plastic embedding material of sample and sample,
As described above, this may change the material character (for example, the key by rupturing in plastic polymer) of plastic embedding material, therefore
Make cutting sample more difficult and lead to chatter and/or inconsistent slice thickness.It has been found that 30 nanometers is for subtracting
The suitable slice thickness of little this effect.Therefore, in SBEM application, microtome as herein described is sufficiently accurate, makes
Obtaining it is not to limit the factor that slice thickness reduces.In the case that microtome is used in optical microscope, it is possible to use with electricity
Hold the equally little slice thickness of the resolution of sensor.
The part of microtome as herein described can be by appointing in inclusion aluminum, titanium and/or stainless various suitable material
A kind of what manufacture.Microtome can include thermocouple with the temperature of monitoring component, thus mitigating heat effect as needed.Send out
Existing, above-mentioned microtome can have weight less than 1kg, the height of the overall width of 235mm, the depth of 73mm and 75mm, and
Can be configured to significantly more less expensive than known commercially available microtome.The slightly rearrangement that the element of microtome can be carried out is cut with adjusting
The size of piece machine, for example, to adapt to specific SEM vacuum chamber.Based on its weight, this microtome can be cut than known to other
Piece machine is more easily installed in scanning electron microscope.Based on its overall dimension, microtome can easily coordinate in scanning electricity
The microscopical 30cm vacuum of son is indoor.This microtome is also compatible with optical microscope, and can be by nonmagnetic substance (example
As aluminum, platinum) manufacture, to prevent or reduce the interference in the magnetic field to SEM under immersion lenses operator scheme.
Fig. 2 D diagram is using the illustrative methods 250 of microtome 100.252, microtome 100 may be mounted at scanning electricity
In the microscopical vacuum chamber of son.254, can prepare sample for study and sample is arranged on microtome 100.256,
Using scanning electron microscope, the exposed surface of sample can be imaged.258, can arrange under the feedback control and keep knife
Piece height, and piezo-activator 146 can be activated to cause the vibration of blade.260, sample can translate across blade with
Remove the thin part of sample, and the vibration activating to suspend blade of piezo-activator 146 can be interrupted.262, permissible
Blade is made to retract away from sample upwards.264, sample can be translated under the electron beam of scanning electron microscope that to return to it former
Beginning image space.
Once sample has returned to image space, due to removing the top of sample by blade, so the exposure table of sample
Face is in the position more slightly lower than the imaging for step 256.Therefore, in sample after 264 return to image space, can adjust
Whole system is compensating this change.This adjustment can be passed through again to focus on newly the electron beam of scanning electron microscope in 266A
Exposed surface realizing.Alternatively (or in addition), this adjustment can be passed through in translation scan ultramicroscope in 266B
Horizontalization platform to be realized so that the exposed surface of sample is in and the imaging identical position for step 256 with moving up sample
Put.268, it is possible to use scanning electron microscope is imaged to the exposed surface of sample, and this process can repeat, Zhi Daoyi
Collect enough data.
In some cases, when sample translates across blade, fragment is likely to accumulate on blade, thus reducing its precision
And concordance, and/or it is retained on the exposed surface of sample, thus potentially introducing error into the image that surface is shot
In.In order to prevent fragment from accumulating on blade 116, one piece of polystyrene foam plastics or other suitable materials can be installed to
Sample 136 pedestal 134 below, i.e. make sample 136 when sample is in image space between material and blade 116.Cause
This, bar 132 can towards actuator 110 mobile so that sample 136 is cut, and bar 132 can move further so that
Material blade 116 remains in any of the above fragment to remove.
If exposed surface scanning electron microscope image display fragment be present on surface, can take steps from
Surface removes fragment, and then can to obtain more complete image.In order to remove on the exposed surface remaining in sample 136
Fragment, sample 136 for example can move through blade 116 for the second time so that blade 116 can clear away any fragment leaves sample
136 face.This can be by being maintained at blade 116 and initial cut identical height, or by first will be remote for blade 116
To complete so that the extra section of sample 136 will not by mistake be removed from the mobile a small distance of the exposed surface of sample 136.
Fig. 3 A and Fig. 3 B is to remove, from sample top, the section that thickness is 40 nanometers by using microtome as above,
The scanning electron microscope image of the continuous exposed surface of tissue sample producing during serial block Surface scan electron microscopy.
For some embodiments it is also possible to remove relatively thin section, in such as little as 15 nanometers of slice, such as Figure 10 A and Figure 10 B
Shown.Fig. 3 C is the image of the calculating difference between pictorial image 3A and the consecutive image of Fig. 3 B.In fig. 3 c, darker area instruction
Smaller difference between consecutive image, and brighter areas indicates the larger difference between consecutive image.As shown in FIG. 3 C, even
Continuous image is largely similar, and this shows the thickness of the section that the structure of tissue sample only removes from sample top in microtome
On somewhat change, reflect microtome remove sample very little part ability.Also as shown in FIG. 3 C, consecutive image it
Between difference relatively uniform in the gamut of image, this show from sample remove part thickness on the surface of the samples
Similarly consistent.These results show that microtome as herein described removes the very thin part of sample with high consistency
Ability, for example, does not have blade to skip the surface (sometimes referred to as " chatter ") of sample.
Fig. 3 D is and Fig. 3 B identical photomicrography image.Fig. 3 D and Fig. 3 E is the continuously sudden and violent of sample generated as above
The scanning electron microscope image on dew surface.Fig. 3 F is the figure of the calculating difference between pictorial image 3D and the consecutive image of Fig. 3 E
Picture.Fig. 3 G is and Fig. 3 E identical photomicrography image.Fig. 3 G and Fig. 3 H is the continuous exposure table of sample generated as above
The scanning electron microscope image in face.Fig. 3 I is the image of the calculating difference between pictorial image 3G and the consecutive image of Fig. 3 H.Figure
3J is and Fig. 3 H identical photomicrography image.Fig. 3 J and Fig. 3 K is sweeping of the continuous exposed surface of sample generated as above
Retouch electron microscope image.Fig. 3 L is the image of the calculating difference between pictorial image 3J and the consecutive image of Fig. 3 K.Fig. 3 M be with
Fig. 3 K identical photomicrography image.Fig. 3 M and Fig. 3 N is the scanning electron of the continuous exposed surface of sample generated as above
MIcrosope image.Fig. 3 O is the image of the calculating difference between pictorial image 3M and the consecutive image of Fig. 3 N.Fig. 3 F, Fig. 3 I, Fig. 3 L
With the result shown in Fig. 3 O with shown in Fig. 3 C and result as above is suitable.
Fig. 4 A is to illustrate, when 14 mobile tissue samples pass through blade, to carry out the vertical position of measurement microtome blade
Multiple data sequence of above-mentioned capacitance sensor in output curve chart.Fig. 4 B is the data of Fig. 4 A illustrating to overlap each other
The curve chart of sequence.As shown in Fig. 4 A to Fig. 4 B, the y-axis of curve chart illustrates the height of blade, and the x-axis diagram of curve chart
Incremental time when sample moves through blade.With reference to the data sequence 402 of Fig. 4 A, can be seen that blade initially quilt 440
It is reduced to desired position.Once blade is positioned as needed, sample can move through blade.The data being given in Fig. 4 A
Display, in the first six time, sample moves through blade, as shown in data sequence 402,404,406,408,410 and 412, sample
Not with blade contact.
With reference to the data sequence 414 of Fig. 4 A, 450 as can be seen that sample contacts blade when it moves through.Sample and
The starting of contact between blade leads to blade to be pushed upwardly, and feedback circuit has an opportunity to correct blade and make blade downward afterwards
Return to desired position.Data sequence 414 with further reference to Fig. 4 A is it can be seen that 460, sample stops and blade contact.
The stopping of the contact between sample and blade leads to blade to come off from the end of sample, and feedback circuit has an opportunity to correct blade afterwards
And make blade back up into desired position.In contact start blade be pushed upwardly, feedback circuit correction so that blade to
Lower movement, contact stop when blade decline and feedback circuit correct so that the feature mode that blade moves up can be whole
See in individual remaining data sequence 416,418,420,422,424,426 and 428.
This data provides better than the obvious advantage being obtained using existing microtome.For example, in known microtome, do not have
Have and determine, for accurate, the straightforward procedure that blade is with respect to the position of sample.Therefore, in many cases, preparing should for SBEM
Microtome needs the surface imaging to sample for the researcher, makes blade move through sample, the surface re-imaging to sample,
And whether any part comparing two images to determine sample is removed.It is removed without part, then blade will move
Dynamic little distance is with closer to sample, and will repeat this process.Repeat this process, until by comparing consecutive image, determining
A part for sample is removed.This process possibly takes and poorly efficient and easy error very much, and must quilt
It is frequently performed, because the period not using microtome of any prolongation may make the accurate relative position of blade and sample unknown.
Additionally, this process introduces big electron dose to sample, this may change the material character of plastic coating (for example, by fracture
Key in plastic polymer), hence in so that cutting sample is more difficult and leads to chatter and/or inconsistent slice thickness.
Therefore, microtome as herein described provides calibration and interrupts the more efficient and effective method recovered.
Fig. 4 B illustrating the data sequence of Fig. 4 A overlapping each other illustrates the concordance of data sequence, and therefore illustrates
A series of concordance of the performance of microtome when microtome removes part from the surface of sample.
In some cases, scanning electron microscope can become to the region on no more than 50 microns × 50 microns of surface
Picture.However, often can have the size in the range of 500 microns × 500 microns using the sample of SBEM technical research.Cause
This, in some cases, the multiple scanning electron microscope images shooting at grade are spliced together to produce sample
The bigger composograph on product surface.For example, as shown in Figure 5, scanning electron microscope can be used for shooting in primary importance
First image of the first area 501 of the exposed surface of sample.Then using scanning electron microscope built-in platform by sample
Move to the second position from primary importance, and scanning electron microscope can be used for shooting the second image of second area 502.
Then can for example using the poster processing soft by two image mosaic together.In some cases, shown in the first image
The region of sample can have the region overlapping with the region of the sample shown in the second image, and overlapping region 503 is permissible
It is used for two image mosaic together to form composograph by the poster processing soft.
Once having had been taken by two images, sample can be returned to by it with the top of sample removed as described above
Primary importance, and can be by coming the first area 504 with overlapping region 506 and second area 505 imaging to newly sudden and violent
This process is repeated on the surface of dew.Can to the first area 507 of the 3rd exposed surface, second area 508 and overlapping region 509,
And the first area 510 to the 4th exposed surface, second area 511 and overlapping region 512 repeat this process.
In order that this technology is combined with SBEM process effectively, platform preferably has and is enough to ensure that sample rests on phase
With position for the precision to each continuous exposed surface imaging.Additionally, between first area 501 and second area 502
Overlapping region 503 is preferably sufficiently large, to guarantee, in the case of some errors translating in view of microtome, there is at least some
Overlapping region is to allow by the first image and the second image mosaic together.Therefore, more accurate platform can allow less
Overlapping region and therefore allow bigger composograph.Based on the built-in platform of typical scanning electron microscope, send out
Existing, about 1 micron of overlapping region is enough in many cases.
It has been found that the built-in platform of FEI NanoSEM 450 scanning electron microscope has enough precision to realize
These purposes.However, if it find that the built-in platform of scanning electron microscope is not accurate, then can install on built-in platform
Center linear translates platform, and microtome can be arranged on halfpace.One suitable halfpace is commercially available
P-625.2XY piezotable from Physik Instrument.In this case, built-in platform can provide coarse adjustment and
Halfpace can provide fine setting.
Fig. 6 A is the scanning electron microscope image of the tissue sample shooting at the operating distance of 3.1mm.Fig. 6 B be
The scanning electron microscope image of the homologue's sample shooting at the operating distance of 4.0mm.Fig. 6 C is the working distance in 4.9mm
The scanning electron microscope image of the homologue's sample shooting from place.Generally speaking, the image shown in Fig. 6 A to Fig. 6 C is said
Understand by reducing operating distance, it is possible to obtain the higher signal-to-noise ratio image of tissue sample.This illustrates above-mentioned microtome
One advantage is, it can allow to shoot scanning electron microscope image at the operating distance more shorter than existing microtome.
It is provided that having the electron beam of the accelerating potential of 2kV in typical scanning electron microscope, and make sample
It is grounded as mentioned above.In this case, 2kV is almost by the electronics that detector is collected, or is about in some cases
1.9kV.Fig. 7 diagram wherein voltage is applied to the replacement microtome 700 of sample 702.This can pass through sample 702 and pedestal
704 electric isolution and the such as Gold plated Layer by being coated on sample 702 realize to sample 702 applied voltage.This can also
By provide insulator 706 electrically insulate pedestal 704, and for example by 708 applied voltages come to pedestal 704 applied voltage
To realize.In this case, apply a voltage to pedestal 704 and can produce electric field line 710.
From the pole piece outgoing of scanning electron microscope and the beam axis along scanning electron microscope is directed to Fig. 7 diagram
The electron beam 712 of charged species 702, and collect secondary and backscattered electron detector 714 from sample 702.Have at one
It is provided that having the electron beam 712 of the accelerating potential of 6kV in body embodiment, and can provide there is -4kV voltage
Sample 702.In this case, electron beam 712 due to bundle slow down and with above-mentioned ground connection sample situation identical energy (2kV)
Collide with sample 702, but due to accelerate (beamacceleration), the energy close to 6kV is had by the electronics that detector 714 is collected, or one
It is about 5.9kV in the case of a little, and greater number of electronics is directed back into detector 714.Therefore, the reality shown in Fig. 7
The data applying scheme collection can have higher quality, and can have the signal to noise ratio of improvement.
In some embodiments, it is possible to use electron tomography is imaged and to improve result.Electron tomography imaging is wherein electric
Beamlet is directed into the technology of sample with the various swings around its center.Fig. 8 A to Fig. 8 C illustrates to be applied to electron tomography
As the microtome 800 of scanning electron microscope of application and the different configurations of detector 802.Fig. 8 A illustrates to be in cutting of 0 ° of inclination
Piece machine 800 and the detector 802 being located at sample 804 top.Fig. 8 B illustrates to be in the microtome 800 of 20 ° of inclinations and is located at sample
The detector 802 of 804 tops.Fig. 8 C illustrates to be in the microtome 800 of 20 ° of inclinations and is located at the side of sample 804 to increase electricity
The detector 802 of sub- detection efficiency.20 ° are illustrated as example slant angle, and actually this angle can be in wide scope
Change in proper angle.
The detailed three dimensional structure collecting permission reconstruction sample 804 of gained electronics.One advantage of this technology is it
The three dimensional structure of study sample 804 in more detail can be allowed, or it can allow to carry out using less accurate microtome
Similar detailed research (because not needing equally thin from the material section of the surface of sample 804 removal).Because this technology needs
Want the ability of rotary sample 804, so it can not be used in combination with the known microtome not allowing sample 804 rotation.With regard to
Microtome as herein described, the rotation of sample 804 can be by the platform of scanning electron microscope (and therefore whole microtome
800) rotation, or realized around the rotation of pivot bearings by pedestal.
Fig. 9 A to Fig. 9 E illustrates another exemplary microtome 900.Microtome 900 includes describing above for microtome 100
Many parts.Microtome 900 includes blade support member 904 is connected to the vertical bender element 902 of linear actuatorss 906.
Vertical bender element 902 is favourable, because it minimizes the vertical motion of blade 908 when blade 908 vibrates back and forth.Section
Machine 900 also includes the insulator 910 for electrically insulating sample 914.Insulator 910 can be by polyether-ether-ketone (PEEK) or other are true
Empty compatible material is made.Microtome 900 also includes slit 912, and polystyrene foam plastics channel washer is (not shown) to be located at slit
To allow cleaning blade 908 after cutting sample 914 in 912.Microtome 900 also includes radiator 916.When radiator 916
For, when in vacuum chamber, radiator 916 provides the extra radiating from linear actuatorss 140.
Fig. 9 B illustrates microtome 900 from another angle.Fig. 9 C is illustrated as the microtome 900 in configuration, wherein sample
Grade is under the electron beam of scanning electron microscope.Microtome 900 in Fig. 9 D diagram cutting configuration, wherein blade 908 cuts
Sample 914.Microtome 900 in Fig. 9 E diagram cleaning configuration, wherein can be clear with the polystyrene foam plastics in slit 912
Clean bar cleaning blade 908.
Figure 10 A and Figure 10 B is to remove, from sample, the section that thickness is 15 nanometers by using microtome as herein described,
The scanning electron microscope image of the exposed surface of tissue sample producing during serial block Surface scan electron microscopy.Figure 10 A
XY view by a heap data is shown, and Figure 10 B illustrates the XZ view by a heap data.
Figure 11 illustrates another exemplary microtome 1000.Microtome 1000 includes being permitted above for microtome 900 description
Multi-part.Microtome 1000 includes blade support member 1004 is connected to the vertical bender element 1002 of linear actuatorss 1006.
Vertical bender element 1002 is favourable, because it minimizes the vertical motion of blade 1008 when blade 1008 vibrates back and forth.
Microtome 1000 also includes the insulator 1010 for electrically insulating sample 1014.Insulator 1010 can be by polyether-ether-ketone (PEEK)
Or other vacuum-compatible materials make.Microtome 1000 also includes slit 1012, and polystyrene foam plastics channel washer (is not schemed
Show) may be located in slit 1012 to allow cleaning blade 1008 after cutting sample 1014.Microtome 1000 also includes dissipating
Hot device 1016.When radiator 1016 is used in vacuum chamber, radiator 1016 provides the extra radiating from linear actuatorss.
Microtome 1000 also includes linear stage or piezotable 1020, capacitance sensor is reset to the top of its scope
Portion.Capacitance sensor can have about 600 microns of scope.After cutting through 600 microns of tissue, linear stage 1020 is permissible
Sensor is increased to the top of capacitance sensor scope, and does not change the position that knife 1008 is with respect to sample 1014.This is effective
Cut coverage along Z axis is increased to the scope (for example, to about 25mm) of linear stage 1020 by ground from 600 microns.
Microtome 1000 can also include the polymer support 1022 positioned at sample 1014 side, and Faraday cup can be installed
Or be fixed on polymer support 1022.The interpolation of support 1022 can allow by using SEM platform accidentally under electron beam
Translate Faraday cup to monitor electron beam current.
Microtome 1000 can also include clamping 1024, to make pedestal remains stationary (for example, therefore pedestal when installing sample
Will not by mistake pivot when installing sample).Once data collection starts it is possible to remove pincers 1024.
The purpose describing for this, this document describes some aspects of the embodiment of present disclosure, advantage and novelty
Feature.Disclosed methods, devices and systems should not be construed as being limiting.On the contrary, present disclosure list
Be related to solely and in various combinations with one another and sub-portfolio all novel and non-aobvious of various disclosed embodiments and
The feature being clear to and aspect.Methods, devices and systems are not limited to any particular aspects or feature or a combination thereof, disclosed enforcement
Scheme also must not be sought survival in any one or more specific advantages or solve problem.
In conjunction with the feature of certain aspects of the present disclosure, embodiment or embodiment description, integer, characteristic, compound, chemistry
Part or group are interpreted as being applied to any other aspect as herein described, embodiment or embodiment, unless with its not phase
Hold.All features disclosed in this specification (including any claims, summary and accompanying drawing), and/or as the displosure
Any method or all steps of process may be combined with any combinations, except in these features and/or step extremely
Some mutually exclusive combinations few.The invention is not restricted to the details of any foregoing embodiments.The present invention extends to this specification
Any novel one of the feature disclosed in (including any claims, summary and accompanying drawing) or any novel group
Close, or any novel one of the step of so disclosed any method or process or the combination of any novelty.
Although assuming the operation that order in a particular order describes the certain methods in disclosed method for convenience,
It is understood that this describing mode includes rearranging, unless language-specific needs particular sorted.For example, retouch in order
The operation stated can be rearranged in some cases or execute simultaneously.Additionally, for simplicity, accompanying drawing may not illustrate
The various modes that disclosed method can be used in combination with additive method.As it is used herein, term " " and " at least one
Individual " include one or more elements specified.That is, if there is two element-specific, then one of these elements there is also,
And therefore there is " one " element.Term " multiple " and " a plurality of " refer to specify two or more in element.
As it is used herein, element list the term "and/or" using between latter two refer to listed
Any one or more in element.For example, phrase " A, B and/or C " refer to " A ", " B ", " C ", " A and B ", " A and C ", " B and
C " or " A, B and C ".
As it is used herein, term " connection " typically refer to physically, magnetically, chemically, electricity ground or with its other party
Formula connection or link, and be not precluded within there is no specific phase irony speech in the case of there is intermediary element between connection element.
In view of the possible embodiment of many of principle disclosed herein can be applied it should be appreciated that shown enforcement
Scheme is only embodiment, and be not construed as to scope of the present disclosure restriction.On the contrary, scope of the present disclosure
At least broad as the present invention being defined by the following claims.It is therefore desirable to protection falls into the scope of these claim
Interior all inventions.
Claims (32)
1. a kind of microtome of a thin part at the top for removing sample, comprises：
Substrate；
Pedestal, is connected to described substrate so that described pedestal can move to cutting position from image space, wherein said pedestal
Having can be in the exposed surface of described sample installed above；
Blade, is connected to described substrate so that when described pedestal is in described image space, can pass through perpendicular to described
The side of the described exposed surface of pedestal moves up described blade to adjust the blade position with respect to described substrate, with selectivity
Ground changes the distance between described blade and described substrate；
Wherein said cutting position is than described image space closer to described blade.
2. microtome according to claim 1, wherein said microtome is arranged on fluorescence microscope, described fluorescence microscopy
Mirror is configured to the surface imaging using camera to described sample.
3. microtome according to claim 1, wherein said microtome is arranged on cathodoluminescence microscope, described negative electrode
Illuminating microscope is configured to the surface imaging using camera to described sample.
4. microtome according to claim 1, wherein said microtome is arranged on photoelectric emission microscope, described
Photoelectric emission microscope is configured to the surface imaging using camera to described sample.
5. microtome according to any one of claim 1 to 4, wherein said blade is arranged on the first computer controls
On linear actuatorss, the linear actuatorss of described first computer controls are connected to described substrate.
6. microtome according to claim 5, wherein said pedestal is arranged on bar, and described bar is coupled by pivot bearings
To described substrate.
7. microtome according to claim 6, wherein by be connected to described substrate second computer control linear
The actuating of actuator, described pedestal can rotate around described pivot bearings.
8. microtome according to claim 7, wherein said first actuator is configured to receive control signal to guide
The motion of described blade, described microtome comprises further：
Sensor, is connected to described first actuator and is configured to produce the described blade position with respect to described substrate for the instruction
The output signal put；And
Computer program, is configured to receive the described output signal from described sensor, is based at least partially on from institute
The described output signal stating sensor produces described control signal, and described control signal is transferred to described first actuating
Device.
9. microtome according to any one of claim 1 to 8, comprises the sample being positioned on described pedestal further.
10. microtome according to claim 9, wherein can select scanning electron to show from the scope of available work distance
Operating distance between the described sample of micro mirror and pole piece.
11. microtomes according to claim 9 or claim 10, wherein voltage is applied to described sample.
12. microtomes according to claim 11, wherein said sample and described microtome electric isolution.
13. microtomes according to any one of claim 1 to 12, wherein said blade is the vibration Buddha's warrior attendant that piezoelectricity controls
Stone blade.
14. microtomes according to any one of claim 1 to 13, comprise the meter including processor and memorizer further
Calculate device, described memory storage is used for combining the calculating to create the three dimensional representation of described sample for the multiple images of described sample
Machine readable instruction.
15. microtomes according to any one of claim 1 to 14, comprise linear piezoelectric platform further, described linear
Piezotable is configured to reset capacitance sensor and does not change the position that described blade is with respect to described substrate.
16. microtomes according to any one of claim 1 to 15, comprise polymer support further, described polymer
Support is connected to described pedestal and is configured to fixing Faraday cup.
17. microtomes according to any one of claim 1 to 16, comprise to clamp, described pincers are connected to described base further
Seat and described substrate, and be configured to, when sample is installed to described pedestal, make described pedestal keep with respect to described substrate
Static.
A kind of 18. microtomes being configured to be arranged in scanning electron microscope, comprise：
It is connected to the blade of actuator, wherein said actuator is connected to the platform of described scanning electron microscope so that described
Actuator can move described knife with respect to described platform on the direction of the beam axis parallel to described scanning electron microscope
Piece, and wherein said actuator is configured to reception control signal to guide the motion of described blade；
Sensor, is connected to described actuator and is configured to produce the output letter with respect to the blade position of substrate for the instruction
Number；And
Computer program, is configured to receive the described output signal from described sensor, is based at least partially on from institute
The described output signal stating sensor produces control signal, and described control signal is transferred to described actuator.
19. microtomes according to claim 18, comprise pedestal further, described pedestal be connected to described platform so that
Described pedestal can move to the cutting position leaving described beam axis by the image space from described beam axis, wherein said cuts
Cut position than described image space closer to described blade.
A kind of 20. methods, comprise：
By Sample location at the image space on the microtome in scanning electron microscope, wherein said image space is described
On the beam axis of scanning electron microscope；
The first exposed surface imaging to described sample；
The height of the blade of described microtome is set；
Described sample is moved to cutting position from described image space, wherein said cutting position more leans on than described image space
Closely described blade and not on described beam axis；
Mobile described sample passes through described blade to remove a part for described sample and to expose the second exposure table of described sample
Face；
Described sample is moved to described image space；And
Described second exposed surface imaging to described sample.
21. methods according to claim 20, after the described height further contained in the described blade of setting, in feedback
Control the lower described height keeping described blade.
22. methods according to claim 20 or claim 21, further contained in described second exposed surface it
Front imaging, by the Electron Beam Focusing of described scanning electron microscope at described second exposed surface.
23. methods according to any one of claim 20 to 22, become further contained in described second exposed surface
As before, along sample described in described beam axial adjustment.
24. methods according to claim 23, further contained in along after sample described in described beam axial adjustment, incite somebody to action
The Electron Beam Focusing of described scanning electron microscope is at described second exposed surface.
25. methods according to any one of claim 20 to 24, wherein the first exposed surface imaging to described sample
Action comprise：
Capture multiple composition images of described first exposed surface using described scanning electron microscope；And
By the plurality of composition image mosaic together to form the composograph of described first exposed surface.
26. methods according to any one of claim 20 to 25, wherein said method comprises to make described section further
Machine tilts so that described beam axis is not orthogonal to the described exposed surface of described sample.
27. methods according to any one of claim 20 to 26, wherein said method comprises to make described section further
Machine rotates.
28. methods according to any one of claim 20 to 27, wherein said method comprises to use polystyrene further
Foam plasticss channel washer cleans described blade.
29. methods according to any one of claim 20 to 28, wherein said microtome is arranged on halfpace, and
And described halfpace is arranged on the built-in platform of described scanning electron microscope.
30. methods according to any one of claim 20 to 29, comprise further to reset capacitance sensor and do not change
Knife is with respect to the position of described sample.
31. methods according to any one of claim 20 to 30, comprise for Faraday cup to be attached to polymer further
Support, described polymer support is connected to described pedestal.
32. methods according to any one of claim 20 to 31, comprise for pincers to be fixed on described pedestal and institute further
State between substrate, to make described pedestal when sample is installed to described pedestal with respect to described substrate remains stationary.
Applications Claiming Priority (3)
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| US201461991929P | 2014-05-12 | 2014-05-12 | |
| US61/991,929 | 2014-05-12 | ||
| PCT/US2015/030359 WO2015175525A1 (en) | 2014-05-12 | 2015-05-12 | Miniature serial sectioning microtome for block-face imaging |
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| CN106461515A true CN106461515A (en) | 2017-02-22 |
| CN106461515B CN106461515B (en) | 2019-12-03 |
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| CN201580025001.4A Active CN106461515B (en) | 2014-05-12 | 2015-05-12 | Miniature serial segmentation slicer for the imaging of block face |
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| Country | Link |
|---|---|
| US (1) | US9933339B2 (en) |
| EP (1) | EP3143376B1 (en) |
| JP (1) | JP6829075B2 (en) |
| CN (1) | CN106461515B (en) |
| WO (1) | WO2015175525A1 (en) |
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Also Published As
| Publication number | Publication date |
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| US20170067800A1 (en) | 2017-03-09 |
| EP3143376A4 (en) | 2018-05-30 |
| EP3143376A1 (en) | 2017-03-22 |
| JP2017519200A (en) | 2017-07-13 |
| US9933339B2 (en) | 2018-04-03 |
| EP3143376B1 (en) | 2020-08-05 |
| JP6829075B2 (en) | 2021-02-10 |
| CN106461515B (en) | 2019-12-03 |
| WO2015175525A1 (en) | 2015-11-19 |
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